2.1 Expression and purification of FGF2 and its variants
Recombinant human fibroblast growth factor 2 (FGF2) and its variants
were expressed in the E. coli system as previously reported
(Huang et al. , 2017). The Fast Mutagenesis System Kit (TransGen
Biotech, Beijing, China) was used to introduce mutations into native
FGF2 expression vector in order to generate the variant constructs. BL21
(DE3) competent cells were transformed with the expression vectors and
cultured in LB medium with shaking at 37 °C to an absorbance of 0.5 at
600 nm, then protein expression was induced by addition of 1 mM
isopropyl 1-thio-D-galactopyranoside (IPTG) and continued incubation for
4 h. Cell pellets were lysed in 150 mM NaCl, 10% glycerol, 25 mM
Hepes-NaOH, pH 7.5, and 5 mM EDTA. The cell suspensions were centrifuged
at 12,000 rpm for 30 min at 4 °C, and the soluble lysate fractions
containing FGF2 variants proteins were purified by heparin affinity
column (Hitrap Heparin HP column, 5 ml), followed by further separation
by Superdex 75 10/300 gel filtration column. The purity of FGF2 variants
was confirmed by 12% sodium dodecyl sulfate polyacrylamide gel
electrophoresis (SDS-PAGE). The purified FGF2 variants were concentrated
using Amicon Ultra Centrifugal Filters and the resulting concentrations
were determined by absorbance measurements using a Nanodrop
spectrophotometer. The detailed amino acid sequences of these proteins
were shown in supplemental table 1 .